PROJECT SUMMARY Endothelium lines blood vessels and interconnects all organ systems. However, endothelial cells exhibit a rich diversity in structure and function. Such heterogeneity is apparent between endothelial cells in different organs, in endothelial cells along a single vascular segment within an organ, and between immediately adjacent cells. This program project grant is founded on the overall hypothesis that endothelium lining pulmonary arteries, capillaries, and veins is phenotypically distinct, where each cell type is specialized to fulfill the unique demands of its vascular niche. Specialization among these cells is encoded by discrete organization of second messenger signaling networks. Therefore, in this competitive renewal each of our projects examines the organization and function of endothelial cell signaling networks. We evaluate the origin of second messenger signals, how these signals spread inside of the cell, what limits the lifespan of the signals, and which effector proteins are principally activated by the signals. For the first time we can measure three-dimensional spread of second messengers in realistic cellular geometries over time. Bacteria such as Pseudomonas aeruginosa impair endothelial cell function during the course of infection that culminates in acute lung injury. P. aeruginosa alters the organization and function of second messenger signaling, and in some instances, the bacterium utilizes enzymes that generate second messenger signals during the host- pathogen interaction. P. aeruginosa and its exoenzymes disrupt the endothelial cell barrier, and hence, our projects use this bacterium and its toxins to probe determinants of endothelial heterogeneity and function, including barrier integrity. All projects take advantage of vertically integrated approaches, ranging from the use of cultured cells (e.g. with control for substrate stiffness) to various in situ (e.g. isolated organs, lung slices, isolated blood vessels, and cell-free lung scaffolds) and in vivo preparations. In each of these cases, attention is paid to what is similar, and what is dissimilar, about the respective cell phenotypes. Altogether, projects systematically address three specific aims or objectives, to: (1) identify and test novel molecular mechanisms (e.g. signatures) that are responsible for site-specific endothelial cell function, especially in the microcirculation, (2) determine the importance of these mechanisms in preclinical models of disease, and (3) translate novel therapeutic approaches to preclinical models of disease. Projects are highly interactive. Defining mechanisms that underlie second messenger signaling networks will not only inform us as to the nature of endothelial cell heterogeneity, but provide unprecedented insight into host-pathogen interactions that disrupt the endothelial cell barrier and cause tissue edema. Mechanistic insight into endothelial signal transduction networks will reveal novel therapeutic approaches to improve endothelial dysfunction.